Abstract

Genetic alterations found in carcinomas can alter specific regulatory
pathways and provide a selective growth advantage by activation of
transforming oncogenes. A subset of these genes, including wild-type
alleles of GLI or c-MYC, and
activated alleles of RAS or
β-catenin, exhibit transforming
activity when expressed in diploid epithelial RK3E cells in
vitro. By in vitro transformation of these
cells, the zinc finger protein GKLF/KLF-4 was recently
identified as a novel oncogene. Although GKLF is normally expressed in
superficial, differentiating epithelial cells of the skin, oral mucosa,
and gut, expression is consistently up-regulated in dysplastic
epithelium and in squamous cell carcinoma of the oral cavity. In the
current study, we used in situ hybridization,
Northern blot analysis, and immunohistochemistry to detect GKLF at
various stages of tumor progression in the breast, prostate, and colon.
Overall, expression of GKLF mRNA was detected by in situ
hybridization in 21 of 31 cases (68%) of carcinoma of the breast.
Low-level expression of GKLF mRNA was observed in morphologically
normal (uninvolved) breast epithelium adjacent to tumor cells.
Increased expression was observed in neoplastic cells compared with
adjacent uninvolved epithelium for 14 of 19 cases examined (74%).
Ductal carcinoma in situ exhibited similar expression as
invasive carcinoma, suggesting that GKLF is activated prior to invasion
through the basement membrane. Expression as determined by Northern
blot was increased in most breast tumor cell lines and in immortalized
human mammary epithelial cells when these were compared with
finite-life span human mammary epithelial cells. Alteration of GKLF
expression was confirmed by the use of a novel monoclonal antibody that
detected the protein in normal and neoplastic tissues in a distribution
consistent with localization of the mRNA. In contrast to most breast
tumors, expression of GKLF in tumor cells of colorectal or prostatic
carcinomas was reduced or unaltered compared with normal epithelium.
The results demonstrate that GKLF expression in epithelial compartments
is altered in a tissue-type specific fashion during tumor progression,
and suggest that increased expression of GKLF mRNA and protein may
contribute to the malignant phenotype of breast tumors.

INTRODUCTION

Multiple physiological changes lead to acquisition of the
malignant phenotype. These include self-sufficiency in growth
signaling, insensitivity to growth-inhibitory signals, inhibition of
apoptosis, immortalization, induction of angiogenesis, and the ability
to invade and metastasize
(1, 2)
. Genetic analyses of
inherited predispositions to develop specific carcinomas enabled
isolation of tumor suppressor genes important in both inherited and
sporadic disease
(3)
, and subsequent functional studies
identified certain of these genes as regulators of classical
transforming oncogenes. Thus, alterations in the tumor suppressor
patched (PTC1), or in other molecules that transduce the hedgehog
signal, result in activation of GLI mRNA expression in virtually all
basal cell carcinomas of the skin
(4,
5,
6,
7)
. In contrast,
alterations in the adenomatous polyposis coli (APC) pathway
activate the β-catenin/TCF-4 complex and transcription of
c-MYC and cyclin D1 during colorectal tumor
progression
(8, 9)
. Modulation of the PTC1 and APC
pathways by alteration of the mouse genome provides additional support
of a role for these gene products in specific tumor types
(10,
11,
12,
13,
14)
. Therefore, these pathways exhibit properties of
a gatekeeper, indicating that alteration of the pathway in a specific
tissue is rate-limiting for tumor progression, and that alterations are
found in a large proportion of inherited as well as sporadic tumors
(15, 16)
.

By expression cloning we recently identified the zinc finger protein
GKLF
3
as a novel transforming oncogene when expressed in RK3E cells, a
diploid epithelial cell line derived from primary rat kidney cells and
immortalized with adenovirus E1A
(17)
. cDNA libraries were
prepared using mRNA from human oral squamous cell or breast carcinoma
cell lines, tumor-types not reported to exhibit frequent genetic
alterations that activate well-characterized oncogenes such as
RAS, GLI, or β-catenin(18)
. Retroviral transduction of these libraries into RK3E
cells induced morphologically transformed foci, 11 of which were
subsequently attributed to enforced expression of wild-type human
c-MYC. Two other transformed foci contained independently
derived, wild-type alleles of GKLF. No other genes were
identified in the screen, suggesting that only a select subset of all
oncogenes are able to transform these cells.

Whereas enforced expression of a human wild-type GKLF
transgene in RK3E cells induces morphological transformation in
vitro and tumorigenicity in athymic mice, the doubling time of
GKLF-expressing cells was considerably longer than for RK3E cells (27 h
versus 12 h, respectively; Refs.
17,, 19
).
Similar results were obtained for other oncogenes, including
GLI and c-MYC, as cells expressing these genes
exhibited doubling times of 18 and 19 h, respectively. These
oncogenes may therefore function in epithelial cells by interfering
specifically with contact inhibition rather than by inducing a more
general increase in the rate of cell division.

In support of a role for GKLF as an oncogene, we observed
increased expression of GKLF mRNA during progression of squamous cell
carcinomas of the oropharynx. As demonstrated by mRNA ISH analysis of
surgical specimens, expression in normal epithelial cells is limited to
the differentiating compartment. Expression in dysplastic oral
epithelium is increased overall and is found in all cell layers, and
GKLF is expressed at similar levels in dysplasia and in invasive
carcinoma. These results identified loss of the compartment-specific
pattern of GKLF mRNA expression in epithelium as a candidate mechanism
of tumor progression in oral cancer
(17)
.

GKLF encodes a DNA-binding transcription factor with
functional domains that mediate activation or repression of
transcription
(20,
21,
22)
. GKLF is essential for the barrier
function of skin, because homozygous knockout mice exhibit
morphologically normal skin but die postpartum due to dehydration
(23)
. Transcriptional targets of GKLF that may be relevant
to epithelial differentiation have been preliminarily identified
(23,
24,
25)
. As shown by analysis of normal mouse or human
tissues, GKLF is preferentially expressed in differentiating epithelial
cells of the skin, gut, oral cavity, and thymus
(17, 20, 23, 26, 27)
. In contrast to human oral squamous cell carcinoma,
expression of GKLF mRNA was found to be reduced in mouse models of
intestinal tumorigenesis or hyperproliferation
(28, 29)
.
Independently, analysis of human colorectal mucosa and tumors by SAGE
confirmed the earlier studies in mice
(30, 31)
.
Specifically, mRNA from specimens of normal colonic mucosa generated
GKLF tags at frequencies of 138 or 99 per million SAGE tags, whereas
mRNA from a microdissected tumor generated only 20 tags per million.
These results suggested that GKLF expression is regulated during
neoplastic progression in a tumor type-specific fashion.

Increased expression of specific oncogenes in tumors can result from
genetic alterations and play a causal role in tumor progression.
Alternatively, expression of some oncogenes is cell cycle dependent,
and increased expression can occur as a consequence of increased
proliferation or altered cell cycle occupancy of tumor cells. In
multiple normal tissues as well as in certain cell lines, GKLF
expression is reduced in actively cycling cells compared with
terminally differentiated or growth-arrested cells, and enforced
expression of GKLF in cultured cell lines can retard cell cycle
progression
(26, 32)
. These properties predicted that GKLF
expression might be reduced in tumors, as observed in colorectal
carcinoma. Increased expression in other tumor-types is therefore
somewhat unexpected and may result from specific alterations in the
pathways that regulate GKLF transcription in normal cells.

To better understand the spectrum of tumor-types that exhibit GKLF
activation, we obtained samples of breast carcinoma, colorectal
carcinoma, and prostatic carcinoma and analyzed expression of GKLF in
malignant cells and in adjacent normal-appearing epithelium
(i.e., uninvolved epithelium). The results show that levels
of GKLF mRNA and protein are each up-regulated before invasion in a
majority of cases of breast cancer, but not in tumors of the colorectum
or prostate. In neoplastic lesions of the breast as well as in cultured
mammary epithelial cells in vitro, increased GKLF expression
appears to precede overtly malignant behavior. The potent transforming
activity of GKLF in vitro, the tumor type-specific
activation of expression in vivo, and activation early
during tumor progression identify this oncogene as a potential effector
of tumor progression in the breast.

MATERIALS AND METHODS

Tissue Procurement.

Fresh-frozen and paraffin-embedded samples were obtained through the
Tissue Procurement Core Facility of the University of Alabama at
Birmingham Comprehensive Cancer Center and through the Southern
Division of the Cooperative Human Tissue Network.

mRNA Expression.

ISH was conducted as described
(17)
using sense and
antisense [35S]-labeled riboprobes prepared by
in vitro transcription of a cDNA fragment corresponding to
the 3′ untranslated region of human GKLF. A
GAPDH antisense probe corresponding to bases 366–680
(GenBank accession no. M33197) was synthesized using a
commercially available template (Ambion, Inc., Austin, TX). High
stringency washes were in 0.1 × SSC and 0.1% (v/v)
2-mercaptoethanol at 58°C for GKLF or 68°C for
GAPDH. Slides were coated with emulsion and exposed for 14
days. The number of silver grains/nucleus were counted within
representative areas by two individuals, and a score from 0.0 to 4.0
was recorded. A score of 0.0 indicated only nonspecific background, as
determined using the sense control, and 1.0 corresponded to an average
of four grains/nucleus.

Breast adenocarcinoma cell lines were obtained from the American Type
Culture Collection (Manassus, MD). HMECs were described previously and
were cultured in mammary epithelial basal media (Clonetics Corp.,
Walkersville, MD; Ref.
33
). Extracts were prepared from
exponentially growing cells at 70% confluence, and total RNA isolation
and Northern blot analysis were performed as described
(17)
.

Isolation of an Anti-GKLF Monoclonal Antibody.

The region of the human GKLF cDNA encoding bases 479-1197
(GenBank accession no. AF105036) was cloned into plasmid pET-32a
and expressed in Escherichia coli BL21(DE3) bacteria as a
histidine-tagged protein. Protein was purified from the bacteria
after induction with
isopropyl-1-thio-β-d-galactopyranoside
using a His-Trap Ni-agarose column (Amersham Pharmacia Biotech,
Piscataway, NJ) and eluted with 500 mm imidazole.
Purified protein was used to immunize two mice, and lymphocytes were
fused with murine myeloma cells (PX63-Ag8.653) as described previously
(34)
. Hybridomas that were immunoreactive in an ELISA
assay for the purified antigen were cloned and recloned by limiting
dilution. Positive clones were identified by ELISA, and an IgG1
antibody (αGKLF) was purified from ascites on a protein A affinity
column.

Immunohistochemistry.

Tissues were fixed in neutral buffered formalin and embedded in
paraffin. Deparaffinized tissue sections were incubated with αGKLF at
a concentration of 1.0 μg/ml for 1 h at room temperature, and
processed as described
(35)
. Immunodetection was performed
using a biotinylated secondary antibody, streptavidin-horseradish
peroxidase detection system (Signet Laboratories, Dedham, MA), and the
chromogenic substrate diaminobenzidine (Biogenex, San Ramon, CA).
Sections were counterstained with hematoxylin. Results were scored by
using a 0.0 to 4.0 scoring system where 4.0 corresponds to a saturated
signal
(36)
.

Statistical Analyses.

Paired t tests were used to compare the differences in
expression in breast epithelial cells at various stages of tumor
progression
(37)
. Pearson correlation coefficients were
used to compare results obtained by ISH to those obtained for the same
cases using immunohistochemistry.

RESULTS

GKLF mRNA Expression Is Up-Regulated during Breast Tumor
Progression.

Previously, SAGE analysis of purified normal breast epithelial cells
detected GKLF transcripts at an abundance of 40 tags per million
(31, 38)
, and Northern blot analysis of breast tumor cell
lines revealed the presence of GKLF transcripts
(17)
.
Using sense and antisense [35S]-labeled
riboprobes, we examined the expression of GKLF mRNA in 31 cases of
carcinoma of the breast. Specificity of hybridization was determined by
using the sense probe as a negative control or by hybridization of the
antisense probe to human foreskin, in which GKLF was specifically
detected in suprabasal epithelial cells (not shown).

Expression of GKLF was detected in malignant cells in 21 of 31 cases of
ductal adenocarcinoma (68%, Fig. 1
⇓
, Table 1
⇓
). For several cases that exhibited no detectable expression of GKLF,
prominent expression of the housekeeping gene GAPDH was
observed, indicating that overall mRNA integrity was maintained and
that failure to identify GKLF transcripts may reflect reduced levels of
expression. GKLF expression was increased in malignant cells of 14 of
19 cases that contained adjacent uninvolved epithelium (Fig. 1A)
⇓
. For 7 of these 14 cases, no specific signal was
detected in adjacent uninvolved epithelium. In the other seven cases,
expression was detected in both uninvolved and malignant cells, with
expression of GKLF in malignant cells increased by 3- to 5-fold
compared with uninvolved epithelium. Within tumors, expression of GKLF
was specific to malignant cells, with little or no expression detected
in stromal components (Fig. 1B)
⇓
.

ISH analysis of GKLF mRNA in
carcinoma of the breast. Two distinct cases were analyzed by
applying an antisense (GKLF-AS)[
35S]-labeled RNA probe to sections of
paraffin-embedded (A) or fresh-frozen
(B) surgical material. Brightfield (left)
and darkfield (right) views are shown. Sections were
stained with H&E. Hybridization to a sense control probe resulted in an
average of 0.4 grains/nucleus (not shown). A, two areas
of the same slide are shown, with uninvolved (i.e.,
morphologically normal) breast epithelium (upper plate)
adjacent to an area (lower plate) containing DCIS
(arrowheads) and additional uninvolved tissue
(arrows). B, IDC admixed with cords of
stroma. Scale bars = 160 μm.

GKLF expression in DCIS was not significantly different from invasive
carcinoma, but expression in both lesions was higher than for
uninvolved breast epithelium (Table 1
⇓
, Fig. 2
⇓
). In contrast to results obtained in breast tumors, examination of
several cases of prostatic carcinoma revealed equal or reduced
expression in tumor cells compared with adjacent uninvolved glandular
epithelial cells (Table 1)
⇓
. In summary, the results suggest that GKLF
mRNA expression is activated in approximately two-thirds of breast
carcinomas, and that expression in positive cases is consistently
induced in DCIS before invasion.

Characterization of a GKLF-specific Monoclonal Antibody.

An IgG1 isotype antibody raised against bacterially expressed
GKLF was subsequently referred to as αGKLF. Immunoblot analysis of
GKLF-transformed RK3E cells and control cell lines detected a single
protein species of 55 kDa consistent with the predicted size of the
full-length polypeptide (data not shown). Compared with RK3E cells or
control cell lines transformed by other oncogenes, apparent GKLF
abundance was increased by several-fold in each of two cell lines
transformed by the human expression vector. The epitope recognized by
the antibody may be denaturation-sensitive, as a signal was obtained
only after overnight exposure of autoradiographic film using a standard
chemiluminescence protocol. The antibody was not sufficiently sensitive
to detect GKLF by immunoblot analysis of extracts of human tumor cell
lines that express the endogenous GKLF mRNA.

The cell type- and tumor type-specific patterns of GKLF mRNA expression
were used to examine the specificity of αGKLF in immunohistochemical
assays. These patterns can be summarized as follows. Human GKLF mRNA is
detected by ISH in differentiating cells of oral epithelium, and is
markedly elevated in oral tumors
(17)
. The mRNA is not
detected in morphologically normal basal or parabasal cells,
particularly within epidermal pegs that extend further into the
submucosa. Mouse GKLF mRNA is similarly found to be more highly
expressed in superficial, differentiating cells of the skin and gut,
and is reduced or absent in basal epithelial cells in both tissues
(20, 23, 26)
. In contrast to human oral and breast cancer,
GKLF mRNA expression is reduced in mouse colorectal tumors compared
with normal epithelium
(29)
, and is similarly reduced in
human colorectal cancer as indicated by SAGE
(31)
.

The staining pattern of αGKLF exhibited a strict concordance with
detection of GKLF mRNA (Figs. 3
⇓
and 4
⇓
, Table 2
⇓
). In positive tissues, αGKLF exhibited a mixed nuclear and
cytoplasmic staining pattern. For uninvolved epithelium, DCIS, and
invasive breast carcinoma alike, the average cytoplasmic staining was
1.8- to 2.5-fold greater than nuclear staining, suggesting that
subcellular localization was not altered during breast tumor
progression in any consistent fashion. Cytoplasmic staining was
subsequently used as a more sensitive indicator of overall expression.

In several samples of skin or oral squamous epithelium, αGKLF bound
specifically to differentiating suprabasal epithelial cells (Fig. 3A)
⇓
. Compared with adjacent uninvolved epithelium, staining
was markedly increased in malignant cells for each of several cases of
squamous cell carcinoma with little or no staining of stromal
components of the tumor, as shown previously for the mRNA
(17)
. Likewise, staining was increased in superficial
cells compared with cells deeper within epithelial crypts of the small
bowel (Fig. 3B)
⇓
or the large bowel (Table 2
⇓
;
P = 0.043). In contrast to oral and breast
tumors, staining was reduced in tumor cells compared with adjacent
superficial epithelial cells for each of four cases of human colorectal
adenoma or carcinoma examined (Fig. 3C⇓
, Table 2
⇓
;
P = 0.027).

Expression of GKLF Protein Is Increased during Neoplastic
Progression in the Breast.

Eighteen cases were tested for GKLF expression by
immunohistochemistry (Table 2
⇓
, Fig. 4
⇓
). Nuclear and cytoplasmic staining of normal breast epithelium, DCIS,
and invasive carcinoma were semiquantitatively assessed. Low-level
staining of tumor cells was observed for 6 cases (e.g.,
cytoplasmic staining ranging from 0.20 to 0.85), with 11 cases
exhibiting higher-level staining (e.g., cytoplasmic staining
ranging from 1.00 to 1.75). These results are consistent with detection
of the mRNA in approximately two-thirds of tumors by ISH. For cases
23–31, which were analyzed by both ISH and immunohistochemical
staining, results of the two methods exhibited a close correlation that
reached statistical significance for invasive carcinoma cells
(N = 8; coefficient = 0.77;
P = 0.024). In DCIS, the correlation was
moderate, although the sample number was small (N = 7; coefficient = 0.43). Perhaps because of the
overall lower level of expression in uninvolved tissue, the correlation
was weakest in uninvolved ducts. Minor differences observed for the two
methods may be attributed to differences in sensitivity and
specificity, to false negative results attributable to partial
degradation of mRNA in some surgical samples, or to analysis of
nonserial sections of the same tissue block. As observed in uninvolved
tissue adjacent to tumors, staining was low or undetectable for each of
five cases of reduction mammoplasty (data not shown).

Apparent GKLF expression as determined by nuclear or cytoplasmic
immunostaining was increased in both DCIS and invasive carcinoma
compared with uninvolved ducts (Table 2
⇓
, Fig. 5
⇓
). For morphologically normal ducts, staining of myoepithelial cells was
not significantly different from that of luminal epithelial cells
(P = 0.303, data not shown). However,
staining of neoplastic cells in DCIS was significantly increased
compared with myoepithelial cells within the same ducts
(P = 0.0001), which was consistent with other
studies indicating similarities between tumor cells and luminal
epithelial cells
(39)
.

Analysis of GKLF in Cultured Breast Epithelial Cells.

Northern blot analysis of breast tumor cell lines revealed variable
levels of GKLF expression relative to a tubulin control (Fig. 6)
⇓
. GKLF expression was high in MCF7 and ZR75-1; intermediate in BT474,
BT20, MDAMB361, and SKBR3; and reduced in MDAMB453 and MDAMB231. Thus,
expression in six of eight breast tumor-derived cell lines was
increased relative to 184 cells, an HMEC population of finite life span
derived from normal breast tissue after reduction mammoplasty
(Lane 1). Expression was similarly increased in 184A1 cells
(33)
. These immortalized cells were derived from 184 cells
by treatment with benzo(a)pyrene. They are wild-type for p53
and p105Rb and are anchorage-dependent and
nontumorigenic in animals. The results obtained for breast tumor cell
lines support the conclusion that GKLF expression is up-regulated at
the mRNA level in most breast tumors, whereas activation in 184A1 cells
is consistent with identification of GKLF induction as an early event.

DISCUSSION

Oncogenes such as c-MYC, GLI, and
GKLF function in a regulated fashion in normal epithelium to
control cellular proliferation and differentiation
(5, 6, 8, 23, 40, 41)
. Analysis of well-characterized tumor types such as
colorectal carcinoma and basal cell carcinoma of the skin suggests that
genetic alterations cluster within specific pathways, rather than
within any specific gene, and that these pathways can function as
regulators of oncogene transcription
(42, 43)
. An activity
common to several oncogenes implicated in carcinoma is the ability to
induce transformed foci in the RK3E assay
(17, 44, 45)
.
This assay is highly specific, as foci result from expression of
tumor-derived mutant (but not wild-type) alleles of RAS or
β-catenin (Ref.
45
and data not
shown), and only GKLF and c-MYC were identified
in a large screen
(17)
. The assay also detects a distinct
subset of oncogenes compared with other host cell lines. With the
exception of RAS, the oncogenes that transform RK3E cells do
not induce foci in NIH3T3 cells
(17, 44, 45)
.

GKLF encodes a zinc finger transcription factor of the
GLI-Krüppel family
(46)
and is distinct from many
other oncogenes in that expression in normal tissue is observed in
terminally differentiating epithelial cells. In addition, expression is
induced in association with cell growth-arrest in vitro(26)
. As predicted by these observations, expression in
certain tumor-types is reduced compared with the relevant normal
epithelia. Thus, GKLF expression is reduced in colorectal tumors, a
result supported by multiple approaches including analysis of RNA
extracted from tissues
(29)
, SAGE
(31)
, and
immunohistochemical analysis of human tissues (this work). ISH analysis
of several prostatic tumors likewise indicates that GKLF is expressed
in normal prostatic epithelium, and that expression can be lost during
tumor progression.

In contrast to colorectal and prostatic carcinoma, GKLF expression is
activated in both invasive carcinoma and preinvasive neoplastic lesions
during progression of most breast carcinomas and virtually all
oropharyngeal squamous cell carcinomas. Breast and oral cancers share a
number of additional molecular alterations. Loss-of-function mutations
frequently affect p53 and p16/CDKN2, whereas a smaller proportion of
tumors (5–20%) exhibit gene amplification of c-MYC,
cyclin D1, erbB-family members including the EGF receptor
and erbB-2/HER-2/neu, or others
(47,
48,
49,
50,
51)
. Unlike carcinomas of the GI tract or skin,
neither breast nor oral carcinoma is reported to exhibit frequent
genetic alterations that activate known transforming oncogenes such as
RAS, β-catenin, c-MYC, or
GLI. By analogy with oncogenes in other tumor types,
disruption of the pathways that control GKLF mRNA expression in breast
epithelial cells and in oral mucosa represents a potential mechanism of
tumor initiation or progression in vivo.

The pattern of GKLF expression in normal epithelia may provide clues as
to how GKLF functions in tumor progression. Stratified squamous
epithelium contains at least four functionally distinct compartments
(52, 53)
. The stem cell compartment is composed of cells
within the basal cell layer that exhibit a capacity for self-renewal,
but which rarely divide. The transit-amplifying compartment is composed
of cells within the basal or parabasal cell layers that exhibit rapid
cell division but a reduced capacity for self-renewal. Differentiation
occurs within the prickle cell layer that contains identifiable
desmosomes, leading to the outermost, keratinized superficial layer.
Whereas mechanisms regulating transitions from one compartment to the
next remain poorly understood, c-MYC activation can induce stem cells
to enter the highly proliferative transit-amplifying compartment
(40)
. Because self-renewal and rapid cell division occur
in distinct cell types, the organization of compartments enables the
rapid turnover of epithelial cells while minimizing the possibility of
sustaining permanent genetic damage in stem cells.

The observation that GKLF functions normally in the prickle cell layer
suggests that each of the three compartments (stem cell,
transit-amplifying, and prickle layer) expresses a transforming
activity or a critical function (e.g., self-renewal or
proliferation) that may contribute to the progression of carcinoma.
These compartments appear to be intermingled in dysplastic stratified
squamous epithelium, with prickle layer markers including GKLF
misexpressed in the basal layers, whereas other basal or parabasal
markers are misexpressed in superficial layers. Loss of
compartment-specific patterns of gene expression may result in
coexpression of the properties of several compartments in a single
cell. For example, specific properties of the prickle cell layer, such
as reduced cellular adhesion to basement membranes, altered adhesion to
other cells, and/or loss of the cellular mechanisms that mediate
contact inhibition could confer invasive or metastatic properties to
oral carcinomas.

To better understand the mechanism of transformation, we are
characterizing transcriptional alterations induced by GKLF when
expressed in epithelial cells in vitro. In the future,
identification of upstream regulators of GKLF transcription in
epithelial cells may elucidate the pathways that regulate GKLF and the
mechanism of deregulation of GKLF in specific tumor-types.

Acknowledgments

We gratefully acknowledge Tom Broker and Louise Chow for
assistance with ISH, Martha Stampfer for the gift of mammary epithelial
cells, and Iuri D. Louro and Pintusorn Hansakul for critical reading of
the manuscript.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.